The strength of transmission at individual synapses in the central nervous system is often highly plastic and appears to be tightly regulated. This regulation plays a critical role in processing and storage of information. Furthermore, derangements in the regulation of synaptic strength seem likely to contribute to many neurological and psychological disorders including epilepsy, depression, schizophrenia, and Alzheimer's disease. The long-term goal of this study is to understand the molecular mechanisms that control synaptic transmission in the central nervous system and to learn how these mechanisms are distributed among different types of synapses. The proposed experiments will examine local regulation of autophosphorylation of a prominent brain Ca2+/calmodulin-dependent protein kinase in hippocampal neurons and phosphorylation of its substrate proteins, including the presynaptic vesicle protein, synapsin I, and proteins of the postsynaptic density. An immunocytochemical procedure will be developed for visualizing and quantifying phosphorylation of functionally significant sites on these proteins in situ after pharmacological and/or physiological manipulation of organotypic cultures or acute hippocampal slices. The method will make use of an existing monoclonal antibody that recognizes the CaM kinase only when it is autophosphorylated at a specific functional site and a complementary antisera that recognizes it only when it is not autophosphorylated at that site. Changes in autophosphorylation of the kinase in subcellular compartments will be recorded by this method at various times after inducing post-tetanic potentiation or long-term potentiation. We will raise similar antibodies against sites on synapsin I that are phosphorylated by CaM kinase II. We will use the antibodies to visualize changes in phosphorylation of synapsin I during induced changes in synaptic efficacy. Proteins in postsynaptic densities that are phosphorylated in situ by the CaM kinase, the A-kinase, or the C-kinase will be identified by biochemical labeling methods. These proteins will be purified from the postsynaptic density fraction, and the sites on the proteins that are phosphorylated in situ will be sequenced in preparation for immunocytochemical studies of their phosphorylation during induced changes in synaptic efficacy. The proposed immunocytochemical method will provide data about the kinetics of individual regulatory phosphorylation reactions and the sequence of those reactions at defined locations within neurons. Such data will permit testing of predictions made by explicit models of the organization of regulatory pathways in different types of synapses.
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